Some aspects of the biogenesis of tRNA from primary gene transcripts to their final functional form in both E. coli and certain eucaryotes will be investigated. In particular, RNase P, essential for the processing of the 5' termini of tRNAs, will be purified and extensively characterized in terms of its interaction with precursor molecules. Studies of tRNA structure-function relationships will also be undertaken through an investigation of both UGA suppressing and non-temperature sensitive ochre suppressing E. coli tRNAI Tyr su oc ion A2. In addition, the nature of the defect in certain other mutants of tRNAI Tyr, which do not produce sufficient quantities of gene transcript for RNA sequence analysis, will be studied by DNA sequence analysis of DNA restriction enzyme fragment containing the appropriate tRNA gene sequences. An attempt will also be made to answer the question: are the promoter sequences utilized by the E. coli tRNA Tyr genes also used by other tRNA genes? Both wild type and genetically altered E. coli tRNA precursor molecules and/or RNase P will be used respectively as substrates and enzyme in studies of RNase-tRNA precursor interactions. Radiochemically pure tRNA precursor species from B. mori will serve as substrate for studies of eucaryotic processing enzymology. RNA nucleotide sequence analysis of Maxam-Gilbert DNA sequence analysis will be used to elucidate the nature of defects altering suppression or processing defects in mutants of tRNA Tyr. DNA restriction enzyme digests of T4, phi 80, E. coli and yeast DNA will be probed by the nitro-cellulose membrane filter blotting and hybridization technique with gene fragments containing the tRNA Tyr promoter elements to determine if these elements are used in the transcriptional control of other tRNA genes.